Ischemia/stroke presents a major health problem worldwide. Since cerebral ischemia is a severe form of metabolic stress that interferes with all major biochemical and molecular biological pathways, it is difficult to identify the disturbances that are directly involved in the pathological process culminating in neuronal cell death. Genetic approaches provide promising new tools to verify whether proteins believed to play a role in this pathological process are indeed key contributors. Gene silencing by small interfering RNAs (siRNAs) can be used to establish whether eliminating the respective gene product aggravates or blocks the pathological process resulting in neuronal cell death. To avoid possible side effects that could be associated with permanently expressed siRNAs, conditional gene silencing approaches have been established where transcription of the silencing RNA sequence is modified by external drugs such as tetracycline. Since these systems rely on drugs potentially producing various side effects that could significantly modify the cellular stress response and thus complicate the interpretation of result, new systems of gene silencing that do not require activation by external chemicals are urgently required. The proposed project focuses on two new systems of conditional gene silencing using promoters activated by endogenous transcription factors or neuronal cell-specific polymerase II promoters as inducers. Our specific aims are to develop new systems of conditional gene silencing, based on the micro RNA (miRNA) approach that will target neurons specifically or control expression of the designed miRNA sequence by promoters that are activated in the course of the pathological process under investigation. To guarantee a neuron-specific silencing effect, we will de- sign constructs where expression of the miRNA sequence is controlled by a neuron-specific promoter. To guarantee stress inducibility, we will use a heat shock promoter or hypoxia-inducible promoter to induce expression of the designed miRNA sequence after ischemia or other severe forms of stress known to be associated with activation of heat shock factors and/or the hypoxia-inducible factor. Using these constructs, we will be able to silence expression of genes coding for apoptotic proteins in a conditional manner. Apoptosis is an active process requiring expression of genes coding for apoptosis-inducing proteins. Post-ischemic expression of these genes could be suppressed efficiently using the new conditional miRNA- based approach. We will produce lentivirus with our most potent constructs, infect primary neuronal cells and expose cultures to transient oxygen/glucose deprivation or hypoxia to test the efficacy of the systems developed. The new conditional gene silencing systems will be invaluable tools for the elucidation of mechanisms of ischemic cell death and could also be exploited in various areas of basic and applied research, including cancer research. PUBLIC HEALTH RELEVANCE: There is urgent need to better understand the pathological process triggered by ischemia/stroke and culminating in neuronal cell death. Conditional gene expression or gene silencing are state-of-the-art tools for investigating mechanisms of ischemic cell death. This proposal focuses on developing two new systems of conditional gene silencing using stress-inducible or neuronal cell specific Pol II promoters. The new conditional gene silencing systems will be invaluable tools for the elucidation of mechanisms of ischemic cell death and could also be exploited in various areas of basic and applied research, including cancer re- search.